The goal of this proposal is to develop the commercial potential of a cell-based assay to screen for compounds with antiviral activity against filoviruses such as Ebola and Marburg viruses. The basis of this bioassay, which has been prototyped with respiratory syncytial virus, is infection-independent expression of a reporter gene from an artificial viral genome present within the cytoplasm of transformed cells. Filoviruses include some of the most notorious human pathogens and cause significant morbidity and mortality due to severe hemorrhagic fever syndromes. Unfortunately, very few therapeutic agents are available to treat infections with these viruses. Efforts to find new antiviral agents will require methods that are amenable to highthroughput assays. Since all viruses are obligate intracellular microbes, any bioassay that determines the effect of various agents on viral replication, must be based on cell culture. This involves working with infectious virus, which is obviously not feasible for agents such as EBOV and MBGV. Significant advances have been made in identifying the cis-acting elements and trans-acting factors involved in replication of many pathogenic negative-strand RNA viruses, including these filoviruses. We plan to develop infection-independent Ebola and Marburg virus minigenome-expression systems in which the expression of an easily measurable enzyme is dependent on the RNA replication and transcription factors that are essential for viral replication. We plan to use these systems in cell-based assays for screening antiviral compounds.